Arrangement for the detection of illumination radiation in a laser scanning microscope

ABSTRACT

An arrangement for the detection of the illumination radiation in a laser scanning microscope, wherein a portion of the illumination radiation is coupled out and detected at the main color splitter, wherein light transmitted through the main color splitter is advantageously detected and/or a portion of the illumination radiation is coupled out and detected before coupling into a light-conducting fiber and/or a portion of the illumination radiation is coupled out and detected at a beam splitter arranged downstream of a light-conducting fiber.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority of German Application No. 103 32 064.4,filed Jul. 11, 2003, the complete disclosure of which is herebyincorporated by reference.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The invention relates to an arrangement for the detection ofillumination radiation in a laser scanning microscope.

b) Description of the Related Art

In DE 197 02 753, a portion of the excitation output is deflected to amonitor diode in a laser scanning microscope in the excitation beam pathin front of the main color splitter by means of an additional reflectingarrangement.

The arrangement for reflection onto the monitor diode can accordingly bearranged in the beam path in such a way that the laser light strikes thecoating or layer in a p-polarized manner while the main color splitteris struck with s-polarization.

The light exiting from the light-conducting fiber is linearly polarizedat better than 100:1. This is achieved by stress-induced birefringence.In a corresponding manner, the linearly polarized laser light can beguided in the fiber in two axes perpendicular to one another. Derivedfrom this degree of polarization, the electric field strength vector hasan x-component and a y-component (s-pole and p-pole) with an intensityratio of 100:1. In order to achieve this degree of polarization at thefiber output, it is necessary to couple in the linearly polarized laserlight parallel to one of the birefringent axes of the fiber. Theorientation of the birefringent axes can vary by fractions of angulardegrees relative to the polarization direction of the laser due toenvironmental influences. The portion of laser light that is coupledinto the respective axes can vary in a corresponding manner. Withrespect to the polarization at the fiber output, this means that theintensity ratio between the x- and y-components of the electrical fieldstrength vector varies. The dichroic layers used for the beam deflectionin the illumination beam path of the LSM preferentially reflect one ofthe two components of the field strength vector (e.g., the x-component).When a monitor diode is arranged in such a way that, in contrast to theillumination beam path, the y-component is preferentially reflected inthe path in which the monitor diode is positioned, there are differentintensity variations in the two beam paths.

Further, a loss in illumination energy results from the additionalelement for reflecting onto the monitor diode.

OBJECT AND SUMMARY OF THE INVENTION

It is the object of the invention to obtain a comparison signal for theexcitation radiation which is correlated to the detector signal in themost optimal manner possible.

This object is met in an arrangement for the detection of theillumination radiation in a laser scanning microscope comprising that aportion of the illumination radiation is coupled out and detected at themain color splitter.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described more fully with reference to a schematicdrawing in FIG. 1. In this connection, reference is had to thedescription of a LSM beam path in DE 19702753 A1.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A laser module LM contains a plurality of lasers L1-L3 which arecombined or unified by means of beam splitters ST1 and are coupled intoa light-conducting fiber F1 by an AOTF and a coupling location K.

Alternatively, coupling can also be carried out in another lightguide F2by means of another beam splitter.

Displaceable collimating optics KO are provided in the illuminationmodule BM. The laser light travels in the direction of the main colorsplitter HFT of the microscope by means of these displaceablecollimating optics KO and beam splitters ST2 and in the direction of thesample, not shown, via an X/Y scanner SC and an objective O. Thedetection beam path perpendicular to the illumination beam path is notshown.

According to the invention, the positioning of a monitor diode iscarried out, alternatively or simultaneously, in a plurality ofadvantageous arrangements:

A: In the illumination direction behind the main color splitter HFT. Thelaser light impinging on a monitor diode MD1 through HFT corresponds tothe proportion not reflected by the HFT. The advantage is that noadditional optical element is required for directing laser light to themonitor diode MD1. Accordingly, light with identical polarization ispresent in the beam path of the monitor diode as well as in theexcitation beam path. Further, a neutral splitter 80/20 or anothersplitting ratio which transmits greater than 50% (or 80% in case of the80/20 splitter) of the incident energy which accordingly reaches themain splitter is selectively used as main color splitter HFT during themeasurement.

Due to the high proportion of transmitted output, the transmitted outputis to a great extent independent from changes in transmission caused bythe effects of temperature (single layer construction). Further, in thisway, the proportion of illumination energy reaching the monitor diode issubject to the same variations as the laser light reflected to thesample. A wavelength can be measured at any time.

B: In the illumination module BM behind the beam splitter ST2,associated with the respective laser port, as is illustrated by way ofexample by the monitor diode MD2. This has the advantage that a parallelmeasurement of a wavelength can be carried out from every laser port. Itis ensured by means of additional blocking filters SF that every monitordiode detects only light from the laser port associated with it.

C: On the laser module LM, for example, behind the AOTF and in front ofthe fiber coupling, advantageously by means of reflection at an uncoatedglass plate GL onto a monitor diode MD3. In this way, it is possible tomeasure the laser output in front of the fiber coupling.

The following measurement modes are advantageously possible for thedifferent arrangements:

I. The monitor diode signal is continuously read out (presetelectronics). This means that averaging takes place over a determinedtime window. Since the AOTF is controlled differently (e.g., theexcitation output is switched off during a return scan) depending on thescanning program (unidirectional, bidirectional, ROI), a mode in whichthe AOTF is constantly switched on must be set for measuring the monitordiode signal. This ensures that the signal measured at the monitor diodeis not dependent upon the scanning program. A measuring processaccordingly comprises the following steps:

-   -   a) the scanning process is stopped    -   b) neutral splitter 80/20 is moved in    -   c) AOTF is switched to continuous mode; only the laser line to        be measured is activated.

Uses in applications:

-   -   a) When a deviation from a reference value/previous measured        value is determined, the excitation output can be corrected by        adjusting the AOTF.    -    (Amplitude of the acoustic wave)    -   b) In connection with the REUSE function (adjustment of        receiving parameters of a stored image) of the software and in        connection with a calibration of the monitor diode, the laser        output for every laser line in the experiment can be adjusted in        an exactly reproducible manner.

II. It is also conceivable to read out the monitor diode signalsynchronous with the scanning process and control of the AOTF. A readoutwould then be possible during the scanning process and a permanentcontrol of the excitation output can be realized.

In order to carry out a measurement of individual lines in this case inan experiment with a plurality of excitation laser lines, a filter wheelwith line selection filters can be arranged in front of the monitordiode. This enables constant recording of the intensity of theindividual lines for a wavelength or a wavelength region.

A spectral splitting of the collinearly superimposed laser radiation ofa plurality of laser light sources in the beam path in front of themonitor diode and spectrally selective detection are also possible.

In order to realize a construction which is as compact as possible andrequires the least space, the monitor diode is adapted to the lightoutput of the respective laser line by adapting the amplification(logarithmic amplification/damping) to the subsequent electronics.

The arrangements according to the invention can advantageously be usedfor:

-   -   1. Stabilization/recording of the excitation output in long-term        experiments    -   (arrangements A and B)    -   2. Quantitative measurement of the excitation output (power        meter function)    -   (arrangements A and B)    -   3. Error search for servicing (coupling efficiency of        light-conducting fiber)    -   (arrangements A, B and C)    -   4. Automatic fiber alignment by two monitor diodes    -   (arrangements A, B and C)

Additional clarification with respect to items 3 and 4: In connectionwith the monitor diode on the laser module and in comparison of the twomonitor diode signals (laser module/illumination module), it is possibleto distinguish between variations in laser output (long-termmeasurement) and misaligned or defective fiber coupling (short-termmeasurement). This makes possible a remote diagnosis by means of theremote capability of the system and, accordingly, an efficientelimination of errors. Items 3 and 4 make use of the presence of monitordiodes behind the light-conducting fibers. This makes it possible, forexample, to check the quality of the fiber input-coupling and can beused in connection with a mechanization of the adjusting elements of thefiber coupling for an automatic adjustment of the fiber coupling.

While the foregoing description and drawings represent the presentinvention, it will be obvious to those skilled in the art that variouschanges may be made therein without departing from the true spirit andscope of the present invention.

1-9. (cancelled).
 10. An arrangement for the detection of theillumination radiation in a laser scanning microscope, comprising that aportion of the illumination radiation is coupled out and detected at themain color splitter.
 11. The arrangement according to claim 10, whereinlight transmitted through the main color splitter is detected.
 12. Anarrangement for the detection of the illumination radiation in a laserscanning microscope, wherein a portion of the illumination radiation iscoupled out and detected before coupling into a light-conducting fiber.13. The arrangement according to claim 12, wherein coupling out iscarried out behind an AOTF arranged in the illumination beam path. 14.An arrangement for the detection of the illumination radiation in alaser scanning microscope, comprising that a portion of the illuminationradiation is coupled out and detected at a beam splitter arrangeddownstream of the light-conducting fiber.
 15. The arrangement accordingto claim 14, wherein light transmitted through the input-couplingsplitter is detected.
 16. A method for the operation of an arrangementaccording to claim 10, comprising the step of carrying out a measurementduring a continuous AOTF mode.
 17. The method for the operation of anarrangement according to claim 10, comprising the step of carrying outthe measurement so as to be synchronous to the line-by-line detection ofthe sample.
 18. The method for the operation of an arrangement accordingto claim 10, comprising the step of carrying out a measurement with aplurality of receivers for diagnosing intensity variations and/ormisalignment.